Project Summary The first synapse of the visual system?between photoreceptors and bipolar cells?is critical to visual processing but displays abnormal specificity and maintenance in the setting of photoreceptor dysfunction. Functional restoration, for example with gene or cell-based therapies, will depend upon a greater understanding of altered synaptic connectivity in human retinal disease. The human retinal organoid system permits the detailed study of synaptic development and maintenance in retinal tissue in a scalable and experimentally accessible fashion. The proposed experiments will use human retinal organoids to address the hypothesis that rod bipolar cell (RBC) dendrites initially form non-specific contacts with rods and cones, which then become increasingly specific during development, and that synaptic promiscuity in retinal diseases represents a recapitulation of these non-specific interactions in early retinal development. This hypothesis will be tested in three Specific Aims. Aim 1 determines the long-term developmental stages of photoreceptor-bipolar cell synaptogenesis and specifically examines whether rod-RBC synaptic specificity is achieved by pruning inappropriate contacts. Aim 2 investigates the short-term dynamics of RBC synaptogenesis in live organoids using a CRISPR-engineered fluorescent reporter line to ascertain whether exploratory dendrite behavior facilitates synaptic specificity. Aim 3 examines the role of glutamate release in synapse formation, maintenance, and specificity as mediated by Cav1.4, the presynaptic calcium channel implicated in X-linked congenital stationary night blindness. The proposed work will provide opportunities to make significant advances in understanding synaptic wiring in human retinal tissue, thus establishing this system to identify molecular targets that maintain or restore synaptic connectivity. This application constitutes the foundation of my career development award (K08) as an academic pediatric retina specialist with very high motivation and institutional support to develop an independent laboratory research program in pediatric retinal disease. In pursuit of these aims, I will be mentored by renowned scientists at Children's Hospital Los Angeles and the University of Southern California, as I gain the necessary skills in human retinal organoid techniques, retinal and synaptic neuroscience, and live cell imaging to achieve independence through R01 funding. Ultimately, my work will explore for the first time human retinal synaptic connectivity during development and establish a research program focused on restoring these connections in the human disease state.